Many chronic/recurrent human infectious disease states are recognized to involve bacterial biofilms. These include urinary tract infections (UTIs), chronic skin infections, otitis media and cystic fibrosis lung infections. In each, bacterial communities form, encased in an extracellular matrix where they are shielded from host immune mechanisms and from the action of currently used antibiotics. This greatly complicates the clearance of these organisms and the ability to cure these infections. Amyloid fibers called curli are critical in the formation of a biofilm extracellular matrix by uropathogenic Escherichia coli (UPEC). This fiber is made up of the major subunit CsgA, and a minor subunit CsgB. Using Thioflavin T fluorescence, circular dichroism and atomic force microscopy the in vitro mechanism by which CsgB nucleates CsgA fibrillization has been elucidated. The methods used in these studies will serve as a model for examining interactions between other components of the curli system. In vivo, the formation of curli amyloid fibers is directed by specific chaperone-like proteins (CsgE and CsgF) to occur extracellularly at the CsgG assembly site in the outer membrane. These proteins also serve to restrict premature, intracellular amyloid fibrillization, which otherwise would be toxic. In this proposal we utilize a combination of biophysical, biochemical, structural, genetic and chemical genetic methodologies to elucidate the structure, function and mechanism of action of the chaperone-like proteins CsgE, CsgF and the outer membrane assembly protein CsgG in facilitating the CsgB-templated fibrillization of CsgA. These studies will elucidate basic principles of curli formation and more generally amyloid formation. Then, using a thiazole ring-fused 2-pyridone scaffold we will investigate the mechanism of action of small molecule inhibitors (curlicides) of curli assembly and curli-mediated biofilm formation. Curlicides will be used as molecular scalpels to dissect the details of the protein-protein interactions required for curli biogenesis. Curli promote biotic and abiotic surface colonization, stabilize cell-cell contacts allowing cell aggregation and thickening of the biofilm layer and confer resistance to the biofilm against environmental stresses and biocides. Thus, curlicides will be tested for their efficacy in preventing the formation of UPEC biofilms on silicone catheter tubing both in vitro and in a murine model of catheter- associated urinary tract infection. This work will provide key insights into processes necessary for the formation of medically important biofilms and will serve as a model to better understand amyloid plaques important in neurodegenerative disorders.